In spite of nearly 40 years research on fibronectin (FN), there are two important structural questions about the FN matrix that are unanswered. The first is, what are the bonds that connect FN molecules to make the matrix fibrils? Our first aim is to develop a new approach to identify these FN-FN bonds. We propose to make FN dimers in which 4-6 highly specific 3C and/or TEV protease sites are inserted between certain domains. These FNs will be used to assemble an FN matrix in cell culture. The matrix will be crosslinked to stabilize binding partners, then cleaved with protease. The FN will be released as small fragments, crosslinked to their bonded partners. These will be identified by binding specificity and antibodies. The precise binding sites will be determined by mass spec. The second question is elasticity. An intriguing mechanical feature of FN fibrils is that they can be stretche 4-fold, making them perhaps the most elastic protein polymer known. The mechanism of the elasticity is controversial. One group concludes that stretching involves unfolding and extending FNIII domains. We believe the stretching involves a conformational change of the FN dimers from compact to elongated, without domain unfolding. We propose two approaches to settle the controversy. (1) We have designed FRET constructs using GFP acceptor and chemical fluorophore donors attached to engineered cys in the domain to be tested. The FRET will report the extent of domain unfolding. (2) We will develop shrec, a super-resolution, single-molecule light microscopy technique, to determine the separation of red and green fluorophores in the first FNIII domains in the dimer. Shrec should have a resolution of ~10 nm, and will tell us whether the FN dimers are in the compact conformation (~20 nm apart), extended conformation (~100 nm), or have unraveled FNIII domains (>100 nm). A third project will investigate a rare kidney disease caused by mutations in FN. We believe that the pathology may be caused by a failure to fold into the compact conformation. We propose to test this by examining the FN dimers using established hydrodynamic techniques. We will also apply the shrec technology discussed above to compare the conformations of wild type and mutant FN at the single molecule level.